Comparative proteomic analysis of glomerular proteins in IgA nephropathy and IgA vasculitis with nephritis.

BACKGROUND: IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN) are related glomerular diseases characterized by marked similarities in immunological and histological findings. We herein performed a comparative proteomic analysis of glomerular proteins in IgAN and IgAVN. METHODS: We used renal biopsy specimens from 6 IgAN patients without nephrotic syndrome (NS) (IgAN-I subgroup), 6 IgAN patients with NS (IgAN-II subgroup), 6 IgAVN patients with 0-8.0% of glomeruli with crescent formation (IgAVN-I subgroup), 6 IgAVN patients with 21.2-44.8% of glomeruli with crescent formation (IgAVN-II subgroup), 9 IgAVN patients without NS (IgAVN-III subgroup), 3 IgAVN patients with NS (IgAN-IV subgroup), and 5 control cases. Proteins were extracted from laser microdissected glomeruli and analyzed using mass spectrometry. The relative abundance of proteins was compared between groups. An immunohistochemical validation study was also performed. RESULTS: More than 850 proteins with high confidence were identified. A principal component analysis revealed a clear separation between IgAN and IgAVN patients and control cases. In further analyses, 546 proteins that were matched with ≥ 2 peptides were selected. The levels of immunoglobulins (IgA, IgG, and IgM), complements (C3, C4A, C5, and C9), complement factor H-related proteins (CFHR) 1 and 5, vitronectin, fibrinogen chains, and transforming growth factor-ß inducible gene-h3 were higher (> 2.6 fold) in the IgAN and IgAVN subgroups than in the control group, whereas hornerin levels were lower (< 0.3 fold). Furthermore, C9 and CFHR1 levels were significantly higher in the IgAN group than in the IgAVN group. The abundance of some podocyte-associated proteins and glomerular basement membrane (GBM) proteins was significantly less in the IgAN-II subgroup than in the IgAN-I subgroup as well as in the IgAVN-IV subgroup than in the IgAVN-III subgroup. Among the IgAN and IgAVN subgroups, talin 1 was not detected in the IgAN-II subgroup. This result was supported by immunohistochemical findings. CONCLUSIONS: The present results suggest shared molecular mechanisms for glomerular injury in IgAN and IgAVN, except for enhanced glomerular complement activation in IgAN. Differences in the protein abundance of podocyte-associated and GBM proteins between IgAN and IgAVN patients with and without NS may be associated with the severity of proteinuria.

IL-6 Generated from Human Hematopoietic Stem and Progenitor Cells through TLR4 Signaling Promotes Emergency Granulopoiesis by Regulating Transcription Factor Expression.

Emergency granulopoiesis, also known as demand-adapted granulopoiesis, is defined as the response of an organism to systemic bacterial infections, and it results in neutrophil mobilization from reservoir pools and increased myelopoiesis in the bone marrow. Indirect and direct initiating mechanisms of emergency granulopoiesis have been hypothesized. However, the detailed mechanism of hyperactive myelopoiesis in the bone marrow, which leads to granulocyte left shift, remains unknown. In this study, we report that TLR4 is expressed on granulo-monocytic progenitors, as well as mobilized human peripheral blood CD34+ cells, which account for 0.2% of monocytes in peripheral blood, and ∼ 10% in bone marrow. LPS, a component of Gram-negative bacteria that results in a systemic bacterial infection, induces the differentiation of peripheral blood CD34+ cells into myelocytes and monocytes in vitro via the TLR4 signaling pathway. Moreover, CD34+ cells directly responded to LPS stimulation by activating the MAPK and NF-κB signaling pathways, and they produced IL-6 that promotes emergency granulopoiesis by phosphorylating C/EBPα and C/EBPß, and this effect was suppressed by the action of an IL-6 receptor inhibitor. This work supports the finding that TLR is expressed on human hematopoietic stem and progenitor cells, and it provides evidence that human hematopoietic stem and progenitor cells can directly sense pathogens and produce cytokines exerting autocrine and/or paracrine effects, thereby promoting differentiation.

Downregulation of miR-26 promotes invasion and metastasis via targeting interleukin-22 in cutaneous T-cell lymphoma.

It has been reported that certain microRNAs (miRNA) are associated with the pathogenesis of lymphoma. We have previously demonstrated that histone deacetylase inhibitors restore tumor-suppressive miRNAs, such as miR-16, miR-29, miR-150, and miR-26, in advanced cutaneous T-cell lymphoma (CTCL). Among these, the function of miR-26 remains unclear. In this study, we aimed to reveal the function of miR-26 in CTCL oncogenesis. First, we confirmed that the miR-26 family was markedly dysregulated in CTCL cell lines and primary samples. In vivo analysis using miR-26a-transduced CTCL cells injected into immunodeficient NOG mice demonstrated the significant prolonged survival of the mice, suggesting that the miRNA had a tumor-suppressive function. We performed gene expression assays and identified 12 candidate miR-26 targets, namely RGS13, FAM71F1, OAF, SNX21, CDH2, PTPLB, IL22, DNAJB5, CASZ1, CACNA1C, MYH10, and CNR1. Among these, IL22 was the most likely candidate target because the IL-22-STAT3-CCL20-CCR6 cascade is associated with tumor invasion and metastasis of advanced CTCL. In vitro analysis of IL22 and IL22RA knockdown and miR-26 transduction demonstrated inhibited CTCL cell migration. In particular, IL22 knockdown induced cell apoptosis. Finally, we conducted in vivo inoculation analysis of mice injected with shIL22-transfected CTCL cells, and found no tumor invasion or metastasis in the inoculated mice, although the control mice showed multiple tumor invasions and metastases. These results, along with our previous data, demonstrated that miR-26 is a tumor suppressor that is associated with tumor invasion and the metastasis of advanced CTCL by regulating the IL-22-STAT3-CCL20 cascade. Therefore, a IL-22-targeting therapy could be a novel therapeutic strategy for advanced CTCL.

Comparative proteomic analysis of glomerular proteins in primary and bucillamine-induced membranous nephropathy.

BACKGROUND: Anti-phospholipase A2 receptor autoantibody (PLA2R Ab)-associated membranous nephropathy (MN) is the most common form of primary MN (pMN). On the other hand, bucillamine (BCL), an antirheumatic drug developed in Japan, was reported to cause a rare form of secondary MN (sMN). Between these MN forms, comparative proteomic analysis of glomerular proteins has not been performed. METHODS: We used renal biopsy specimens from 6 patients with PLA2R Ab (+) pMN, 6 patients with PLA2R Ab (‒) pMN, 6 patients with BCL-induced sMN, and 5 control cases (time 0 transplant biopsies). Proteins were extracted from laser-microdissected glomeruli and analyzed using mass spectrometry. The quantification values of protein abundance in each MN group were compared with those in the control group. RESULTS: More than 800 proteins with high confidence were identified. Principal component analysis revealed a different distribution between the pMN and sMN groups. For further analysis, 441 proteins matched with ≥ 3 peptides were selected. Among the pMN and sMN groups, we compared the profiles of several protein groups based on the structural and functional characteristics, such as immunoglobulins, complements, complement-regulating proteins, podocyte-associated proteins, glomerular basement membrane proteins, and several proteins that are known to be associated with kidney diseases, including MN. In all MN groups, increased levels of immunoglobulins (IgG, IgA, and IgM), complements (C3, C4, and C9), complement factor H-related protein 5, type XVIII collagen, calmodulin, polyubiquitin, and ubiquitin ligase were observed. For some proteins, such as type VII collagen and nestin, the fold-change values were significantly different between the pMN and sMN groups. CONCLUSIONS: Between the pMN and BCL-induced sMN groups, we observed common and different alterations in protein levels such as known disease-associated proteins and potential disease marker proteins.

Long-term prognosis of monoclonal immunoglobulin-associated glomerular diseases with non-organized deposits: A report of 38 cases from a Japanese single center.

Monoclonal immunoglobulin (MIg)-associated glomerular diseases with non-organized deposits are rare disorders. They have recently been categorized into light chain deposit disease (LCDD), light and heavy chain deposit disease (LHCDD), heavy chain deposit disease (HCDD), proliferative glomerulonephritis with MIg deposits (PGNMID) and its light chain only variant (PGNMID-LC), and membranous glomerulopathy with light chain-restricted deposits (MG-LC). In our Japanese cohort of more than 9,500 patients who underwent renal biopsy (1979 - 2020), we evaluated clinicopathological features and long-term outcomes in 38 patients with MIg-associated glomerular diseases with non-organized deposits: LCDD (n = 9), LHCDD (n = 8), HCDD (n = 5), PGNMID-membranoproliferative glomerulonephritis (MPGN) (n = 7), PGNMID-LC (n = 2), and MG-LC (n = 7). In patients with LCDD, a low estimated glomerular filtration rate (eGFR) at biopsy, a high detection rate of urinary MIgs, a high incidence rate of multiple myeloma, and sever tubulointerstitial and vascular lesions were significant clinicopathological characteristics. Median duration of follow-up in each group was 42 - 114 months. Most patients were treated with steroid-based therapy. Patients with LCDD, LHCDD, HCDD, and MG-LC were recently treated with bortezomib-based therapy. Renal survival rate was significantly shorter for LCDD than of PGNMID and MG-LC. Patient survival rate was significantly longer for MG-LC than HCDD and PGNMID. Major causes of death were pulmonary and cardiovascular complications. Among disease groups, significant differences were observed in eGFR at biopsy, detection rates of urinary MIgs, incidence rates of multiple myeloma, severities of tubulointerstitial and vascular lesions, and long-term outcomes.

Evaluation of a newly proposed renal risk score for Japanese patients with ANCA-associated glomerulonephritis.

BACKGROUND: We determined the usefulness and prognostic ability of the renal risk score (RRS), proposed in Europe, for Japanese patients with antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (AAGN) and high myeloperoxidase (MPO)-ANCA positivity; these aspects remain to be verified. METHODS: This retrospective study was conducted on 86 Japanese patients with new, biopsy-confirmed AAGN. We calculated the RRS and analyzed the relationship between this classification, and clinicopathological features and prognosis. We also compared the predictive values between RRS for endpoints including renal death and conventional prognostic tools for patients with AAGN. RESULTS: There were 33, 37, and 16 patients in the low-, medium-, and high-risk groups, respectively. All patients were MPO-ANCA positive. The median follow-up period was 33 months; 16 (18.6%) patients progressed to end-stage renal disease (ESRD). In the high-risk group, 9/16 (56.3%) patients progressed to ESRD, and renal prognosis was significantly poorer than that in other groups (low-risk group, P < 0.001; medium-risk group, P = 0.004). In Cox multivariate regression analysis, RRS was an independent, poor renal prognostic factor (hazard ratio 5.22; 95% confidence interval 2.20-12.40; P < 0.001). The receiver-operating characteristic curves of the RRS for each endpoint were comparable with those of the 2010 histological classification and those of the severity classification of Japanese rapidly progressive glomerulonephritis. CONCLUSIONS: This is the first study to report the usefulness of the RRS for predicting renal outcomes among Japanese patients with AAGN. Our predictive value of the RRS was comparable with that of conventional prognostic tools.

VEGF-VEGFR2 inhibitor-associated hyaline occlusive glomerular microangiopathy: a Japanese single-center experience.

BACKGROUND: Inhibitors of vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) signaling, such as bevacizumab (Bmab), are used for the treatment of various advanced cancers. However, these inhibitors induce renal thrombotic microangiopathy (TMA). Recently, two European cohort studies showed a distinctive histopathological pseudothrombotic pattern different from TMA in Bmab-treated patients. METHODS: We analyzed 9 renal biopsies from proteinuric cancer patients treated with VEGF-VEGFR2 inhibitors in our Japanese cohort. Clinical and laboratory features were also assessed in these patients. RESULTS: All 9 patients had moderate to heavy proteinuria with normal or slightly elevated serum creatinine levels. On light microscopy, a patchy pattern of hemispherical/spherical lesions along glomerular capillary walls was a characteristic finding. On immunofluorescence microscopy, staining for immunoglobulins (IgM dominant) at varying intensities was observed mainly along glomerular capillary walls. Especially, hemispherical/spherical positive staining for immunoglobulins was a characteristic pattern. Immunohistochemical studies showed positive staining for immunoglobulins and negative staining for CD61-positive platelets in capillary hemispherical/spherical lesions and positive VEGF staining in podocytes. On electron microscopy, variably electron-dense material in dilated glomerular capillaries and partial effacement of podocyte foot processes were observed. After the withdrawal of VEGF-VEGFR2 inhibitors, proteinuria improved without any specific treatment in 8 patients. CONCLUSIONS: Histopathological findings in our patients treated with VEGF-VEGFR2 inhibitors were consistent with those observed in the recently described new form of Bmab-associated hyaline occlusive glomerular microangiopathy. This form should be considered in proteinuric cancer patients treated with VEGF-VEGFR2 inhibitors. Discontinuing VEGF-VEGFR2 inhibitors may lead to improvement of glomerular microangiopathy induced by these drugs.

Clinicopathological features and outcomes of diabetic kidney disease with extracapillary hypercellularity: a Japanese single-center experience.

BACKGROUND: The prognostic significance of glomerular extracapillary hypercellularity (EXHC) in diabetic kidney disease (DKD) is unclear. The aim of this study was to investigate the clinicopathological features and outcomes of DKD patients with EXHC. METHODS: We studied 70 cases of renal biopsy-confirmed type 2 DKD that were diagnosed between 2004 and 2014 and compared the clinicopathological features and outcomes of 22 patients with EXHC (EXHC group) with those of 48 patients without EXHC (control group). All of the patients were Japanese. We assessed the renal biopsy specimens based on the Renal Pathology Society classification system. Clinical and laboratory data were collected at the time of the renal biopsy, and renal outcomes were assessed based on progression to end-stage renal disease (ESRD) requiring renal replacement therapy. The median duration of the observation period was 3 years. RESULTS: In pathological features, nodular sclerosis (Kimmelstiel-Wilson lesions) was observed more frequently in the EXHC group than in the control group (63.6% vs. 35.4%, P = 0.027). There were no significant intergroup differences in clinical features or renal outcomes. Univariate and multivariate Cox regression analyses of all patients showed that a high level of proteinuria, a low initial eGFR, and severe interstitial inflammation were poor prognostic factors. CONCLUSIONS: EXHC is related to nodular sclerosis, which is a known risk factor for ESRD. Careful observation is needed during the follow-up of DKD patients with EXHC, although there were no significant differences in renal outcomes between the EXHC and control groups.

Comparative proteomic analysis of renal proteins from IgA nephropathy model mice and control mice.

BACKGROUND: High-IgA ddY (HIGA) mice, an animal model of human IgA nephropathy (IgAN), spontaneously develop nephropathy with glomerular IgA deposition and markedly elevated serum IgA levels from 25 weeks of age. METHODS: We performed a comparative proteomic analysis of the renal proteins collected from HIGA mice and control C57BL/6 mice at 5 or 38 weeks of age (the H5, H38, C5, and C38 groups) (n = 4 in each group). Proteins were extracted from the left whole kidney of each mouse and analyzed using nano-liquid chromatography-tandem mass spectrometry. The right kidneys were used for histopathological examinations. RESULTS: Immunohistochemical examinations showed glomerular deposition of IgA and the immunoglobulin joining (J) chain, and increased numbers of interstitial IgA- and J-chain-positive plasma cells in the H38 group. In the proteomic analysis, > 5000 proteins were identified, and 33 proteins with H38/H5 ratios of > 5.0, H38/C38 ratios of > 5.0, and C38/C5 ratios of < 1.5 were selected. Among them, there were various proteins that are known to be involved in human IgAN and/or animal IgAN models. Immunohistochemical examinations validated the proteomic results for some proteins. Furthermore, two proteins that are known to be associated with kidney disease displayed downregulated expression (H38/H5 ratio: 0.01) in the H38 group. CONCLUSIONS: The results of comparative proteomic analysis of renal proteins were consistent with previous histopathological and serological findings obtained in ddY and HIGA mice. Various proteins that are known to be involved in kidney disease, including IgAN, and potential disease marker proteins exhibited markedly altered levels in HIGA mice.

Comparison of measurements of anti-PLA2R antibodies in Japanese patients with membranous nephropathy using in-house and commercial ELISA.

BACKGROUND: The prevalence of antibodies against M-type anti-phospholipase A2 receptor (PLA2R) was reported to be ~ 70-80% in early studies on idiopathic membranous nephropathy (iMN) cohorts from Western countries, China, and Korea, and ~ 50% in recent studies on two Japanese iMN cohorts. METHODS: We developed an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of anti-PLA2R antibodies, and examined sera from 217 patients with iMN, 22 patients with secondary MN (sMN), and 50 healthy individuals. All patients and healthy individuals were Japanese. The relationships between levels of anti-PLA2R antibodies and clinical parameters were analyzed. Serum samples were also tested using a standardized commercial ELISA (Euroimmun, Germany). RESULTS: In our ELISA, OD values greater than the mean + 3 standard deviation of healthy subjects were considered to be positive for anti-PLA2R antibodies. Of the patients with iMN, 33.6% (73/217) were positive, but all sMN patients were negative. Our ELISA and the Euroimmun ELISA had a high concordance (93.5%). The proportion of patients with nephrotic syndrome was significantly higher in anti-PLA2R antibody-positive patients than in antibody-negative patients (65.8 vs. 37.5%, P < 0.001). Levels of anti-PLA2R antibodies were significantly correlated with levels of urinary protein and serum albumin (P = 0.004 and P < 0.001, respectively). CONCLUSIONS: The prevalence of anti-PLA2R antibodies in our Japanese iMN cohort was lower than that in the previous studies from other countries and other Japanese institutes. The low prevalence of antibodies may be related with the characteristics of enrolled patients with mild proteinuria and undetectable antibody levels.

Membranoproliferative glomerulonephritis with unusual deposits of parallel arrangement striated structure: a new pathological entity?
.

A 71-year-old male with a past history of lower limb arteriosclerosis obliterans developed nephrotic syndrome and renal dysfunction. Renal biopsy showed diffuse global endocapillary proliferative lesions with infiltration of mononuclear cells and occasional foam cells. An irregular double contour of the glomerular basement membrane and global mild-to-moderate mesangial proliferative lesions were observed, indicating membranoproliferative glomerulonephritis. Congo red staining was negative. Routine immunofluorescence studies showed no obvious immunoglobulin or complement depositions. Electron microscopy showed endocapillary proliferative lesions and infiltration of macrophages with abundant lysosomes. Irregular subepithelial, subendothelial, and mesangial electron-dense deposits were observed in glomeruli. In these electron-dense deposits, parallel arrangement striated structures were detected. All known disease entities with Congo red-negative and immunoglobulin-negative glomerular deposits were pathologically excluded. The glomerular lesion in our case might be a new disease entity.
.

Analysis of PLA2R1 and HLA-DQA1 sequence variants in Japanese patients with idiopathic and secondary membranous nephropathy.

BACKGROUND: Several recent studies in patients with idiopathic membranous nephropathy (iMN) from Western and Asian counties showed that some single nucleotide polymorphisms (SNPs) within the PLA2R1 and HLA-DQA1 genes are significantly associated with iMN. However, there is only 1 report on analysis of PLA2R1 and HLA regions in Japanese patients with iMN. METHODS: A total of 58 patients with iMN, 26 patients with secondary MN (sMN), and 50 patients with other diseases were enrolled. All patients were Japanese. We selected 6 SNPs within PLA2R1 and 1 SNP within HLA-DQA1, which were significantly associated with iMN in reported white European cohorts, and sequenced these exons using genomic DNA prepared from peripheral mononuclear cells from each patient. We then analyzed differences in PLA2R1 and HLA-DQA1 sequence variants among the 3 groups. RESULTS: Genotypic and allelic frequency distributions for 3 out of 6 SNPs within PLA2R1, rs3749117, rs35771982, and rs2715918 were significantly different between the iMN and control groups. Allelic frequency distributions for SNP rs2187668 within HLA-DQA1 were significantly different between the iMN and control groups. There were no correlations between PLA2R1 and HLA-DQA1 sequence variants and clinical parameters in patients with iMN. There were no significant differences in genotypic or allelic frequency distributions for examined SNPs between the sMN and control groups. CONCLUSIONS: There are some differences in PLA2R1 SNP distributions between previously reported cohorts from other countries and our Japanese cohort of patients with iMN, while there is a significant association between SNP rs35771982 and iMN in most of reported cohorts.

Clinicopathological and long-term prognostic features of membranous nephropathy with crescents: a Japanese single-center experience.

BACKGROUND: Three recent studies from the United States and China reported the clinicopathological features and short-term prognosis in patients with membranous nephropathy (MN) and crescents in the absence of secondary MN, anti-glomerular basement membrane (GBM) antibodies, and anti-neutrophil cytoplasmic antibodies (ANCA). METHODS: We compared clinicopathological and prognostic features in 16 MN patients with crescents (crescent group) and 38 MN patients without crescents (control group), in the absence of secondary MN, anti-GBM antibodies, and ANCA. Median follow-up periods in the crescent and control groups were 79 and 50 months, respectively. RESULTS: Decreased estimated glomerular filtration rates (<50 mL/min/1.73 m2), glomerulosclerosis, and moderate-to-severe interstitial fibrosis were more frequently observed in the crescent group than in the control group (P = 0.043, P = 0.004, and P = 0.035, respectively). Positive staining rates for glomerular IgG2 and IgG4 were significantly different between the 2 groups (P = 0.032, P = 0.006, respectively). Doubling of serum creatinine during follow-up was more frequently observed in the crescent group than in the control group (P = 0.002), although approximately two-thirds of patients in the crescent group were treated with immunosuppressive therapy. Crescent formation and interstitial fibrosis were risks for doubling of serum creatinine [hazard ratio (HR) = 10.506, P = 0.012; HR = 1.140, P = 0.009, respectively]. CONCLUSIONS: This is the first Japanese study demonstrating significant differences in clinicopathological and prognostic features between the 2 groups. Most patients in the crescent group may develop a long-term decline in renal function despite immunosuppressive therapy.

Long-term prognosis of AL and AA renal amyloidosis: a Japanese single-center experience.

BACKGROUND: Few studies have been conducted on the long-term prognosis of patients with amyloid light chain (AL) and amyloid A (AA) renal amyloidosis in the same cohort. METHODS: We retrospectively examined 68 patients with biopsy-proven renal amyloidosis (38 AL and 30 AA). Clinicopathological findings at the diagnosis and follow-up data were evaluated in each patient. We analyzed the relationship between clinicopathological parameters and survival data. RESULTS: Significant differences were observed in several clinicopathological features, such as proteinuria levels, between the AL and AA groups. Among all patients, 84.2 % of the AL group and 93.3 % of the AA group received treatments for the underlying diseases of amyloidosis. During the follow-up period (median 18 months in AL and 61 months in AA), 36.8 % of the AL group and 36.7 % of the AA group developed end-stage renal failure requiring dialysis, while 71.1 % of the AL group and 56.7 % of the AA group died. Patient and renal survivals were significantly longer in the AA group than in the AL group. eGFR of >60 mL/min/1.73 m2 at biopsy and an early histological stage of glomerular amyloid deposition were identified as low-risk factors. A multivariate analysis showed that cardiac amyloidosis and steroid therapy significantly influenced patient and renal survivals. CONCLUSIONS: Our results showed that heart involvement was the major predictor of poor outcomes in renal amyloidosis, and that the prognosis of AA renal amyloidosis was markedly better than that in previously reported cohorts. Therapeutic advances in inflammatory diseases are expected to improve the prognosis of AA amyloidosis.

Hydroxychloroquine binding to cytoplasmic domain of Band 3 in human erythrocytes: Novel mechanistic insights into drug structure, efficacy and toxicity.

Hydroxychloroquine (HCQ) is a widely used drug in the treatment of autoimmune diseases, such as arthritis and systemic lupus erythematosus. It has also been prescribed for the treatment of malaria owing to its lower toxicity compared to its closely related compound chloroquine (CQ). However, the mechanisms of action of HCQ in erythrocytes (which bind preferentially this drug) have not been documented and the reasons underlying the lower side effects of HCQ compared to CQ remain unclear. Here we show that, although the activity of erythrocyte lactate dehydrogenase (LDH), but not GAPDH, was inhibited by both HCQ and CQ in vitro, LDH activity in erythrocytes incubated with 20 mM HCQ was not significantly reduced within 5 h in contrast to CQ did. Using HCQ coupled Sepharose chromatography (HCQ-Sepharose), we identified Band 3, spectrin, ankyrin, protein 4.1R and protein 4.2 as HCQ binding proteins in human erythrocyte plasma membrane. Recombinant cytoplasmic N-terminal 43 kDa domain of Band 3 bound to HCQ-Sepharose and was eluted with 40 mM (but not 20 mM) HCQ. Band 3 transport activity was reduced by only 23% in the presence of 20 mM HCQ. Taken together, these data demonstrate that HCQ binds to the cytoplasmic N-terminal domain of Band 3 in human erythrocytes but does not inhibit dramatically its transport activity. We hypothesize that the trapping of HCQ on Band 3 contributes to the lower side effects of the drug on energy production in erythrocytes.

Enzymatic and thermodynamic profiles of a heterotetramer lactate dehydrogenase isozyme in swine.

Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the final step of glycolysis and produces NAD+. In somatic cells, LDH forms homotetramers and heterotetramers that are encoded by two different genes: LDHA (skeletal muscle type, M) and LDHB (heart type, H). Analysis of LDH isozymes is important for understanding the physiological role of homotetramers and heterotetramers and for optimizing inhibition of their enzymatic activity as it may result in distinct effects. Previously, we reported that hydroxychloroquine (HCQ) inhibited LDH activity, but we did not examine isozyme specificity. In the present study, we isolated heterotetrameric LDH (H2M2) from swine brain, determined its kinetic and thermodynamic properties, and examined the effect of HCQ on its activity compared to homotetrameric LDH isozymes. We show that: (1) the Km values for H2M2-mediated catalysis of pyruvate or lactate were intermediate compared to those for the homotetrameric isozymes, M4 and H4 whereas the Vmax values were similar; (2) the Km and Vmax values for H2M2-mediated catalysis of NADH were not significantly different among LDH isozymes; (3) the values for activation energy and van't Hoff enthalpy changes for pyruvate reduction of H2M2 were intermediate compared to those for the homotetrameric isozymes; (4) the temperature for half residual activity of H2M2 was closer to that for M4 than for H4. We also show that HCQ had different affinities for various LDH isozymes.

Different Expression Patterns of Toll-Like Receptor mRNAs in Blood Mononuclear Cells of IgA Nephropathy and IgA Vasculitis with Nephritis.

Mucosal immunity may play a key role in IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN). IgAVN is characterized by the presence of non-thrombocytopenic palpable purpura, associated with glomerulonephritis with IgA-dominant immune deposits. Recent studies have shown the up-regulation of Toll-like receptors (TLRs) in patients with IgAN or IgAVN. Among TLRs that mediate innate immune reactions, TLR2, TLR4, and TRL5 recognize bacterial components, while TLR3, TLR7, and TLR9 recognize viral components. Here we compared the expression levels of TLR mRNAs in peripheral blood mononuclear cells (PBMCs) from 49 IgAN patients, 20 IgAVN patients, and 20 patients with thin basement membrane nephropathy (TBMN), unrelated to immune-mediated pathogenesis, as a control. The real-time RT-PCR analysis revealed the significantly higher expression levels of TLR2, TLR3, TLR5, TLR7, and TLR9 mRNAs in PBMCs of IgAN and IgAVN patients, compared to TBMN patients. Importantly, TLR4 mRNA levels were significantly higher in IgAN patients than in IgAVN patients, while its expression levels were comparable in IgAVN patients and TBMN patients. In contrast, TLR5 and TLR9 mRNA levels were significantly higher in IgAVN patients than in IgAN patients. In IgAN patients, expression levels of TLR2, TLR3, TLR5, or TLR9 mRNA were correlated with proteinuria levels, and TLR4 mRNA levels were correlated with serum IgA levels. In IgAVN patients, however, there was no such correlation. The up-regulated expression of TLR mRNAs in PBMCs may be related to the development of IgAN and IgAVN. The distinct expression patterns between these two diseases may reflect their different pathogenetic mechanisms.

ATPase activity and ATP-dependent conformational change in the co-chaperone HSP70/HSP90-organizing protein (HOP).

Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change.

Effect of CYP3A5 and ABCB1 polymorphisms on the interaction between tacrolimus and itraconazole in patients with connective tissue disease.

PURPOSE: The aim of this study was to investigate the effect of itraconazole (ITCZ), a potent inhibitor of CYP3A4 and P-glycoprotein, on the blood concentration 12 h after tacrolimus administration (C 12h) in relation to CYP3A5 6986A>G and ABCB1 3435C>T genotype status in patients with connective tissue disease (CTD). METHODS: Eighty-one CTD patients taking tacrolimus (Prograf®) once daily at night (2100 hours) were enrolled in this study. Whole blood samples were collected 12 h after tacrolimus administration at steady state. RESULTS: The dose-adjusted tacrolimus C 12h with or without ITCZ co-administration was significantly higher in patients with CYP3A5*3/*3 than in those with the CYP3A5*1 allele [CYP3A5 *1/*1 vs. *1/*3 vs. *3/*3 = 1.67 vs. 2.70 vs. 4.83 ng/mL/mg (P = 0.003) and 0.68 vs. 0.97 vs. 2.20 ng/mL/mg (P < 0.001), respectively], but differences were not observed for ABCB1 genotypes. However, there was no difference in the increase rate of the dose-adjusted C 12h of tacrolimus between CYP3A5 or ABCB1 genotypes (P = 0.378 and 0.259). On the other hand, reduction of the estimated glomerular filtration rate exhibited a correlation with the C 12h of tacrolimus after ITCZ co-administration (r = -0.482, P = 0.009). CONCLUSIONS: In CYP3A5*3/*3 patients, because the metabolic pathway for tacrolimus occurs only through CYP3A4, the combination with ITCZ seems to lead to a higher risk of acute renal dysfunction. Therefore, we suggest that the target blood tacrolimus concentration be set as low as possible through dose-adjustment for patients with the CYP3A5*3/*3 allele.

Validation of the Japanese histologic classification 2013 of immunoglobulin A nephropathy for prediction of long-term prognosis in a Japanese single-center cohort.

BACKGROUND: A new Japanese histologic classification (JHC) of immunoglobulin A nephropathy (IgAN) for prediction of long-term prognosis was proposed in 2013. The goal of this study was to validate the JHC system in a Japanese single-center cohort. METHODS: A retrospective study was conducted in 198 Japanese adult patients with IgAN. Clinical findings including blood pressure, urinary protein, estimated glomerular filtration rate (eGFR), and outcomes were evaluated in these patients. The glomerular lesion percentage score (GLPS) [number of glomeruli with cellular crescents, fibrocellular crescents, global sclerosis, segmental sclerosis, or fibrous crescents/number of total obtained glomeruli × 100 (%)] was assessed in each patient and categorized into histologic grades (HGs) of HG1 (<25 %), HG2 (25-49 %), and HG3/4 (≥50 %). Associations of GLPS (HG) with disease progression (50 % eGFR decline or end-stage renal disease requiring dialysis) within 10 years after biopsy and the rate of annual eGFR decline were examined. RESULTS: During a median follow-up period of 12.0 years after biopsy, disease progression occurred in 12.8 % (12/94) of HG1 patients, 32.3 % (21/65) of HG2 patients, and 46.2 % (18/39) of HG3/4 patients. The risk of disease progression was significantly higher in the HG2 and HG3/4 groups than in the HG1 group (odds ratios: 3.3 and 5.9 vs. 1). A higher GLPS was significantly associated with a higher risk of disease progression and a greater annual eGFR decline. CONCLUSION: The newly proposed JHC system 2013 based on GLPS (HG) was well correlated with long-term prognosis in our cohort of Japanese adult patients with IgAN.